Oleg Shapiro

Mps1 Mediated Phosphorylation of Hsp90 Confers Renal Cell Carcinoma Sensitivity and Selectivity to Hsp90 Inhibitors

SUMMARY The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors.

Valproic acid decreases urothelial cancer cell proliferation and induces thrombospondin-1 expression.

BackgroundPrevention of bladder cancer recurrence is a central challenge in the management of this highly prevalent disease. The histone deacetylase inhibitor valproic acid (sodium valproate) has anti-angiogenic properties and has been shown to decrease bladder cancer growth in model systems. We have previously shown reduced expression of thrombospondin-1 in a mouse model and in human bladder cancer relative to normal urothelium. We speculated that inhibition of angiogenesis by valproate might be mediated by this anti-angiogenic protein.MethodsBladder cancer cell lines UMUC3 and T24 were treated with valproate or another histone deacetylase inhibitor, vorinostat, in culture for a period of three days. Proliferation was assessed by alamar blue reduction. Gene expression was evaluated by reverse transcription of RNA and quantitative PCR.ResultsProliferation assays showed treatment with valproate or vorinostat decreased proliferation in both cell lines. Histone deacetylase inhibition also increased relative expression of thrombospondin-1 up to 8 fold at 5 mM valproate.ConclusionsHistone deacetylase inhibitors warrant further study for the prevention or treatment of bladder cancer.

The metastatic potential of renal tumors: Influence of histologic subtypes on definition of small renal masses, risk stratification, and future active surveillance protocols

OBJECTIVE The influence of histology in metastatic potential is often overlooked when discussing the management options of small renal masses (SRM), with size or growth rate often serving as the triggers for the intervention. We aim to re-examine the definition of a SRM by evaluating the metastatic potential of renal masses incorporating tumor size and histology to create metastatic risk tables. MATERIALS AND METHODS Surveillance Epidemiology and End Results (SEER)-18 registries database was queried for all cases of clear cell, papillary, and chromophobe renal cell carcinoma (RCC) diagnosed between 2004 and 2012. There were 55,478 cases identified that included 43,783, 8,587, and 3,208 cases of clear cell, papillary, and chromophobe, respectively. Tumors were stratified using 1-cm increments to determine the metastatic potential by calculating the metastatic rate at presentation for different size intervals in histologic categories. RESULTS For all 3 histologies, tumors measuring 5cm or less had a rate of metastatic RCC at presentation of less than 4%. The metastatic potential was highest for clear cell, followed by papillary and then chromophobe tumors. Setting a cutoff of no more than 3% for metastatic potential to be called a SRM, makes clear cell carcinoma and papillary carcinoma a SRM up to 4cm, whereas the chromophobe RCC would be considered a SRM up to 7cm. CONCLUSION Although clinical staging and tumor size have been the key determinants in decision-making of patients with solid renal tumors, the histology-specific risks of metastatic potential are different for each mass. The definition of a SRM should be based on the metastatic potential and not on tumor size alone. This information could be helpful for counseling and managing patients with SRMs as well as for modifying active surveillance protocols.

Chromophobe Renal Cell Carcinoma is the Most Common Nonclear Renal Cell Carcinoma in Young Women: Results from the SEER Database.

PURPOSE The renal cell cancer incidence is relatively low in younger patients, encompassing 3% to 7% of all renal cell cancers. While young patients may have renal tumors due to hereditary syndromes, in some of them sporadic renal cancers develop without any family history or known genetic mutations. Our recent observations from clinical practice have led us to hypothesize that there is a difference in histological distribution in younger patients compared to the older cohort. MATERIALS AND METHODS We queried the SEER (Surveillance, Epidemiology and End Results) 18-registry database for all patients 20 years old or older who were surgically treated for renal cell carcinoma between 2001 and 2008. Patients with unknown race, grade, stage or histology and those with multiple tumors were excluded from study. Four cohorts were created by dividing patients by gender, including 1,202 females and 1,715 males younger than 40 years old, and 18,353 females and 30,891 males 40 years old or older. Chi-square analysis was used to compare histological distributions between the cohorts. RESULTS While clear cell carcinoma was still the most common renal cell cancer subtype across all genders and ages, chromophobe renal cell cancer was the most predominant type of nonclear renal cell cancer histology in young females, representing 62.3% of all nonclear cell renal cell cancers (p <0.0001). In all other groups papillary renal cell cancer remained the most common type of nonclear renal cell cancer. CONCLUSIONS It is possible that hormonal factors or specific pathway dysregulations predispose chromophobe renal cell cancer to develop in younger women. We hope that this work provides some new observations that could lead to further studies of gender and histology specific renal tumorigenesis.

Preoperative cross-sectional imaging allows for avoidance of unnecessary adrenalectomy during RCC surgery

OBJECTIVES To assess the frequency of adrenal involvement and the reliability of preoperative imaging to predict adrenal involvement in patients treated for cortical renal masses at a single institution. METHODS Using a retrospective pathology database, we identified 117 consecutive patients who underwent radical nephrectomy and concomitant ipsilateral adrenalectomy at our institution over the course of 2 decades. Patient demographics, tumor characteristics, and radiographic results were obtained for analysis. RESULTS Of 117 patients, only 6 (5.1%) were identified as having adrenal involvement. The average age of the patient was 58.3 years, and the average tumor size was 7.13 cm. The mean tumor size in patients without adrenal involvement was 6.79 cm, whereas in those with adrenal involvement, it was 9.62 cm (P = 0.057). Of 6 patients with adrenal involvement, 5 had imaging studies available for review, and all 5 demonstrated suspicion for adrenal involvement preoperatively. Among 111 patients without adrenal involvement, 53 (47.7%) had imaging available for review, with only 3 (5.7%) demonstrating suspicion for adrenal involvement. The negative predictive value was 100%, whereas the sensitivity and specificity were 100% and 94.3%, respectively. CONCLUSIONS Ipsilateral adrenal involvement in renal cell carcinoma is uncommon and reliably predicted by preoperative cross-sectional imaging. Among all adrenalectomies in this series, nearly 95% were performed unnecessarily. With careful review, preoperative imaging can help avoid unnecessary adrenalectomy during radical nephrectomy in patients with renal cortical tumors.

Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.

Sporadic renal angiomyolipoma in a patient with Birt-Hogg-Dubé: chaperones in pathogenesis

Birt-Hogg-Dubé (BHD) is an autosomal dominant genetic syndrome caused by germline mutations in the FLCN gene that predisposes patients to develop renal tumors. Renal angiomyolipoma (AML) is not a renal tumor sub-type associated with BHD. AML is, however, a common phenotypic manifestation of Tuberous Sclerosis Complex (TSC) syndrome caused by mutations in either the TSC1 or TSC2 tumor suppressor genes. Previous case reports of renal AML in patients with BHD have speculated on the molecular and clinical overlap of these two syndromes as a result of described involvement of the gene products in the mTOR pathway. Our recent work provided a new molecular link between these two syndromes by identifying FLCN and Tsc2 as clients of the molecular chaperone Hsp90. Folliculin interacting proteins FNIP1/2 and Tsc1 are important for FLCN and Tsc2 stability as new Hsp90 co-chaperones. Here we present a case of sporadic AML as a result of somatic Tsc1/2 loss in a patient with BHD. We further demonstrate that FNIP1 and Tsc1 are capable of compensating for each other in the chaperoning of mutated FLCN tumor suppressor. Our findings demonstrate interconnectivity and compensatory mechanisms between the BHD and TSC pathways.

MP88-13 TUMOR SUPPRESSOR TSC1 IS A NEW HSP90 COCHAPERONE THAT FACILITATES FOLDING OF KINASE AND NON-KINASE CLIENTS

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essen- tial component of the cellular homeostatic machinery in eukary- otes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co- chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilita- tor of Hsp90-mediated folding of kinase and non-kinase clients— including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.

PD46-02 CARCINOMAS OF THE RENAL MEDULLA: A COMPREHENSIVE GENOMIC PROFILING (CGP) STUDY

INTRODUCTION AND OBJECTIVES: BAP1 is a ubiquitin carboxy-terminal hydrolaseenzyme (UCH)whichalongwith theASXL1/2 protein forms the PR-DUB, chromatin associated complex which removes monoubiquitin from H2A in nucleosomes regulating gene expression.BAP1 is a tumor suppressor genecommonlymutatedgene in clear cell renal cell carcinoma (ccRCC) and other cancers such as uveal melanoma (UM) and mesothelioma. In tumors, BAP1 mutations occur throughout the protein well outside the N-terminal UCH catalytic region while C-terminal BAP1 truncations/mutations in the ULD domain also inactivate BAP. The aim of this study was to investigate the potential mechanisms whereby these highly spatially distinct mutations in BAP1, many of which occur outside the UCH catalytic domain, serve to completely inactivate the enzyme function. We focused on BAP1s obligate partner protein ASXL1/2 which binds the BAP1 ULD domain and may exert a global regulatory function in the context of PR-DUB in chromatin. A mechanistic understanding of these events might provide insight towards restoration of normal BAP1 functions in tumors leading to therapeutic benefit. METHODS: BAP1 mutations derived from multiple tumor types were identified, including UM, Mesothelioma and ccRCC and introduced into expression vectors and the proteins expressed as recombinants in e.coli and purified to homogeneity. BAP1 and ASXL1/2 protein complexes were evaluated for protein interaction and UCH enzyme activity using GST-interaction studies, Western blot and gel filtration analysis. Molecular modeling of the complex was performed by Blast analysis, molecular docking methods and refined using insights from our mutagenic study. RESULTS: This structure-function study revealed that the BAP1 and ASXL1/2 is direct, high affinity and that multiple contact points between BAP1, ASXL1/2 exist that stabilize the native catalytic structure the The domain structure of BAP1 is unique, multi-lobed and seems to be highly allosteric with respect to enzyme activation. ASXL1/ 2 is an obligate partner for BAP1 and the enzyme is dead with out ASXL1/2 binding which occurs in the BAP1 ULD domain: many tumor derived mutants in the BAP1 ULD domain disrupt ASXL1/2 binding and abolish BAP1 activity. We also discovered a highly conserved domain in ASXL1/2 as minimally essential for BAP1 binding and activation. Mutations in multiple tumors at widely scattered locations decreased ubiquitin hydrolase activity in re-constituted protein complexes and destabilized binding of BAP1 and ASXL1/2 upon modeling. CONCLUSIONS: Different mutations derived from multiple tumors occur throughout the sequence of BAP1 and apparently act allosterically to alter the ability of ASXL1/2 to both bind and/or interact unproductively with the BAP1 UCH region which leads to loss of enzymatic activity and loss of tumor suppression. This analysis suggests that small molecule approaches might be used to reactivate the latent UCH activity in some tumors and restore wild type function of BAP1.

Comparision of histologic distribution of RCC in young and older patients: Results from the SEER database.

419 Background: Renal cell cancer (RCC) incidence is relatively low in younger patients, encompassing 3-5% of all RCC tumors. These tumors tend to be due to hereditary syndromes and genetic mutations that predispose to cancer development. Patients with hereditary renal cancer (HRC) are at a higher risk of multiple tumors and bilateral disease. We hypothesize that there is a difference in histologic distribution in the younger patients and that the younger distribution contains more aggressive histologic subtypes. METHODS SEER 18-registries database was queried for all patients ≥20 years old that were surgically treated for renal cell carcinoma between the years 2001 and 2008. Patients with unknown race, grade, stage, or histology were excluded from the study. Histologies selected were clear cell, papillary, chromophobe, sarcomatoid, and collecting duct. Three cohorts were created with the ages 20-44, 45-64, and ≥65 year olds that contained 3,926, 19,661, and 16,323 patients respectively. Chi-square analysis was used to compare the histologic distributions between the cohorts. RESULTS There was no difference in the incidence of clear cell RCC between the three cohorts (p = 0.63). The histology distribution was not different in the 45-64 year olds compared to those ≥65 (p = 0.47). The non-clear cell histologies were different between the 3 age groups (p < 0.001). There were a larger percentage of patients in the younger patients that had chromophobe tumors compared to all non-clear cell histologies (p< 0.001). CONCLUSIONS The difference in the non-clear cell histologic distribution between the groups is most likely due to genetic mutations predisposing these patients to chromophobe RCC. There has been limited data on HRCs, due in large part to its low incidence. Although the HRCs are known to have a most common histology, it is likely that this information is incomplete, as younger patients have undiagnosed genetic mutations that led to development of chromophobe tumors. [Table: see text].